U.S. patent number 5,258,513 [Application Number 07/914,583] was granted by the patent office on 1993-11-02 for substituted cyclopropylamino-1,3,5-triazines.
This patent grant is currently assigned to UCB S.A.. Invention is credited to Eric Cossement, Jean Gobert, Solo Goldstein, Berend Jan Van Keulen, Ernst Wulfert.
United States Patent |
5,258,513 |
Van Keulen , et al. |
November 2, 1993 |
Substituted cyclopropylamino-1,3,5-triazines
Abstract
New substituted cyclopropylamino-1,3,5-triazines having the
formula ##STR1## wherein R.sub.1 is C.sub.1 -C.sub.3 -alkyl,
C.sub.3 -C.sub.5 -cycloalkyl or C.sub.1 -C.sub.3 -alkyl-C.sub.3
-C.sub.5 -cycloalkyl R.sub.2 is bis(2-hydroxyethyl)amino,
3-hydroxy-1-azetidinyl, 3-methoxy-1-azetidinyl, 3-oxo-1-azetidinyl,
morpholino, 4-hydroxypiperidino, thiomorpholino, thiomorpholino
S-oxide, thiomorpholino S,S-dioxide, 3-thiazolidinyl,
3-thiazolidinyl S-oxide, 3-thiazolidinyl S,S-dioxide or
8-oxa-3-azabicyclo[3,2,1]oct-3-yl. Processes for the preparation
thereof and pharmaceutical composition containing them are also
given. The compounds are useful for the treatment of disorders
associated with Alzheimer's disease, senile dementia, Alzheimer's
type and with any evolutive cognitive pathology, and also for the
treatment of depression, anxiety, mood disturbances, inflammatory
phenomena and asthma.
Inventors: |
Van Keulen; Berend Jan (Tubize,
BE), Goldstein; Solo (Montreal, CA),
Cossement; Eric (Brussels, BE), Gobert; Jean
(Brussels, BE), Wulfert; Ernst (Brussels,
BE) |
Assignee: |
UCB S.A. (Brussels,
BE)
|
Family
ID: |
10698941 |
Appl.
No.: |
07/914,583 |
Filed: |
July 20, 1992 |
Foreign Application Priority Data
|
|
|
|
|
Jul 25, 1991 [GB] |
|
|
9116039 |
|
Current U.S.
Class: |
544/58.2;
544/207; 544/113; 544/205; 544/105; 544/60; 544/206 |
Current CPC
Class: |
A61P
29/00 (20180101); A61P 25/24 (20180101); A61P
11/06 (20180101); C07D 251/18 (20130101); C07D
411/04 (20130101); A61P 11/00 (20180101); A61P
25/22 (20180101); C07D 417/04 (20130101); A61P
25/20 (20180101); A61P 25/26 (20180101); C07D
401/04 (20130101); A61P 25/28 (20180101); Y10S
514/826 (20130101); Y10S 514/886 (20130101) |
Current International
Class: |
C07D
498/08 (20060101); C07D 498/00 (20060101); C07D
411/04 (20060101); C07D 251/18 (20060101); C07D
251/00 (20060101); C07D 417/00 (20060101); C07D
417/04 (20060101); C07D 411/00 (20060101); C07D
401/04 (20060101); C07D 401/00 (20060101); C07D
251/08 (); C07D 401/04 (); C07D 413/04 (); C07D
417/04 () |
Field of
Search: |
;544/113,205,207,206,60,58.2,105 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Ford; John M.
Attorney, Agent or Firm: Wenderoth, Lind & Ponack
Claims
We claim:
1. A cyclopropylamino-1,3,5-triazine, including its optically
active isomers and racemic mixtures of the formula ##STR12##
wherein R.sub.1 represents an alkyl radical, an unsubstituted
cycloalkyl radical or a cycloalkyl radical substituted by at least
one alkyl radical, the alkyl radicals having 1 to 3 carbon atoms
and the cycloalkyl radicals having 3 to 5 carbon atoms, and
R.sub.2 represents a bis(2-hydroxyethyl)amino,
3-hydroxy-1-azetidinyl, 3-methoxy-1-azetidinyl, 3-oxo-1-azetidinyl,
morpholino, 4-hydroxypiperidino, thiomorpholino, thiomorpholino
S-oxide, thiomorpholino S,S-dioxide, 3-thiazolidinyl,
3-thiazolidinyl S-oxide, 3-thiazolidinyl S,S-dioxide or
8-oxa-3-azabicyclo[3,2,1]oct-3-yl radical,
or a non-toxic pharmaceutically acceptable acid addition salt
thereof.
2. A cyclopropylamino-1,3,5-triazine as claimed in claim 1, wherein
R.sub.2 represents a morpholino, thiomorpholino or thiomorpholino
S,S-dioxide radical or a non-toxic pharmaceutically acceptable acid
addition salt thereof.
3. A compound as claimed in claim 1, namely
2-cyclopropylamino-4-morpholino-6-n-propyl-1,3,5-triazine or a
non-toxic pharmaceutically acceptable acid addition salt
thereof.
4. A compound as claimed in claim 1, namely
2-cyclopropyl-4-cyclopropylamino-6-morpholino-1,3,5-triazine or a
non-toxic pharmaceutically acceptable acid addition salt
thereof.
5. A compound as claimed in claim 1, namely
2-cyclobutyl-4-cyclopropylamino-6-morpholino-1,3,5-triazine or a
non-toxic pharmaceutically acceptable acid addition salt
thereof.
6. A compound as claimed in claim 1, namely
2-cyclopropyl-4-cyclopropylamino-6-thiomorpholino-1,3,5-triazine or
a non-toxic pharmaceutically acceptable acid addition salt
thereof.
7. A compound as claimed in claim 1, namely
2-cyclopropyl-4-cyclopropylamino-6-thiomorpholino-1,3,5-triazine
S,S-dioxide or a non-toxic pharmaceutically acceptable acid
addition salt thereof.
Description
The present invention relates to new substituted
cyclopropylamino-1,3,5-triazines and the non-toxic,
pharmaceutically acceptable acid addition salts thereof, as well as
to processes for the preparation thereof and to the therapeutic use
thereof. It also relates to pharmaceutical compositions containing
these new compounds.
2-trifluoromethyl-1,3,5-triazines which are substituted in the
4-position, inter alia, by an alkyl, substituted or unsubstituted
alkylamino, dialkylamino, cycloalkylamino, morpholino or
4-alkyl-1-piperazinyl radical, and in the 6-position by the same
radicals, except the alkyl radical, are already known from Japanese
Patent Application No. 25786/78. According to this patent
application, these compounds have tranquilizing and anticonvulsive
properties.
Moreover, British Patent No. 1,053,113 describes
1,2-dihydro-1-hydroxy-1,3,5-triazines which are substituted in the
2-position by an imino radical, optionally substituted by an alkyl,
alkenyl, cycloalkyl, phenyl or naphthyl (optionally substituted by
an alkyl radical) radical, in the 4-position by a dialkylamino,
dialkenylamino, N-alkyl-alkenylamino, aziridinyl, azetidinyl,
pyrrolidinyl, piperidino, hexahydroazepinyl, heptamethyleneimino,
octamethyleneimino or morpholino radical, each of said heterocyclic
radicals being possibly substituted by 1 to 3 alkyl radicals, and
in the 6-position by a hydrogen atom or an alkyl, alkenyl,
alkoxyalkyl, cycloalkyl, phenyl or naphthyl radical, optionally
substituted by an alkyl, aralkyl, alkylaralkyl, alkoxyaralkyl or
haloaralkyl radical. According to this patent, these compounds are
antihypertensive agents which can be used for the treatment of
hypertension and shock states; they are also described as secretion
inhibitors and central nervous system depressants. These compounds
are prepared by oxidation of the corresponding 1,3,5-triazines
carrying the same substituents in the 2-, 4- and 6-positions.
However, there is no suggestion in this patent that the
intermediate 1,3,5-triazines could have any pharmacological
activity. Moreover, this patent does not describe any
1,3,5-triazine substituted by a cyclopropylamino radical.
Finally, U.S. Pat. No. 4,956,367 (assigned to the assignee of the
present invention) describes
2-amino-4-morpholino-6-propyl-1,3,5-triazines in which the amino
group in the 2-position is substituted by different radicals such
as, for example, a hydroxyl group or a hydroxyalkyl radical. These
compounds can be used for the treatment of cognitive and behavioral
disorders associated with ageing and with dementia syndromes, for
example, those associated with Alzheimer's disease. However, this
patent does not describe 1,3,5-triazines substituted by a
cyclopropylamino radical.
Continuing its research work in this field, applicant has now found
new cyclopropylamino substituted 1,3,5-triazines which have
valuable pharmaceutical properties and, in particular, the property
to promote learning and to attenuate the amnesic effect resulting
from cholinergic hypofunctioning induced by a cholinergic
antagonist such as, for example, scopolamine. The cholinergic
system is widely involved in the phenomena of memorization and
learning. Thus, for example, administration of an anticholinergic
agent such as scopolamine to young subjects gives rise to memory
deficiencies similar to those observed in elderly subjects.
Conversely, cholinergic agents, such as physostigmine, are capable
of combating the amnesia resulting from the administration of
anticholinergic agents (S. D. GLICK et al., Behavioral Biology, 7,
(1972), 245-254; U. SCHINDLER et al., Drug Develop. Res., 4,
(1984), 567-576). For this reason, the compounds according to the
invention can be used for the treatment of cognitive and behavioral
disorders associated with ageing and with dementia syndromes. In
particular, they are used in the treatment of disorders associated
with Alzheimer's disease, with senile dementia, Alzheimer's type
and with any evolutive cognitive pathology (C. G. GOTTFRIES,
Psychopharmacology, 86, (1985), 245-252; C. G. GOTTFRIES,
Neurobiology of Ageing, 4, (1983), 261-271).
The compounds according to the present invention also have a
central serotonergic activity, demonstrated by the power which they
have to induce a particular stereotypy in rats usually known as
"Wet Dog Shake" (A. R. GREEN and D. J. HEAL in "Neuropharmacology
of Serotonin", Ed. A. R. GREEN, Oxford Univ. Press, 1985, Chapter
12, pages 326 to 365). It is known that serotonin plays an
important role in the regulation of the neuroendocrine function,
which may be disturbed in pathologies such as depression, anxiety
and mood disturbances. A decrease in serotonergic activity is
associated with numerous changes in mood and somatic functions
occurring in depressed patients (H. Y. MELTZER and M. T. LOWY in
"Psychopharmacology: The Third Generation of Progress", Ed. H. Y.
MELTZER, Raven Press, New York, 1987, Chapter 52, pages 513 to
520). The compounds according to the invention can thus be used for
the treatment of these various pathologies associated with a
slowing down in serotonergic activity.
In addition, at the peripheral level, the compounds according to
the invention also have a bronchospamolytic activity and an
inhibiting activity on the release of mastocyte mediators during an
anaphylactic aggression. The compounds according to the invention
moreover potentiate the muscle-relaxing effect of a
.beta.-adrenergic agonist (for example isoprenaline) on a smooth
muscle contracted by histamine, and also have an anti-inflammatory
and anti-edema activity. For this reason, the compounds according
to the invention can also be used in the treatment of inflammatory
phenomena and asthma, in particular as an alternative to treatment
with theophylline or even with bronchodilatory agents such as
.beta.-sympathomimetic agents, which are known to cause, during
prolonged administration, desensitization of the .beta.-adrenergic
receptors in the bronchi, to the extent of rendering the
bronchospasm of chronic asthmatics insensitive and irreversible to
the action of these agents. More particularly, the present
invention relates to new substituted
cyclopropylamino-1,3,5-triazines having the general formula
##STR2## wherein R.sub.1 represents an alkyl radical, an
unsubstituted cycloalkyl radical or a cycloalkyl radical
substituted by at least one alkyl radical, preferably by one or two
alkyl radicals, the alkyl radicals having 1 to 3 carbon atoms and
the cycloalkyl radicals having 3 to 5 carbon atoms, and
R.sub.2 represents a bis(2-hydroxyethyl)amino,
3-hydroxy-1-azetidinyl, 3-methoxy-1-azetidinyl, 3-oxo-1-azetidinyl,
morpholino, 4-hydroxypiperidino, thiomorpholino, thiomorpholino
S-oxide, thiomorpholino S,S-dioxide, 3-thiazolidinyl,
3-thiazolidinyl S-oxide, 3-thiazolidinyl S,S-dioxide or
8-oxa-3-azabicyclo[3,2,1]oct-3-yl radical, and to the non-toxic
pharmaceutically acceptable acid addition salts thereof.
The preferred compounds according to the present invention are the
cyclopropylamino-1,3,5-triazines of general formula I wherein
R.sub.2 represents a morpholino, thiomorpholino or thiomorpholino
S,S-dioxide radical, and the non-toxic pharmaceutically acceptable
acid addition salts thereof.
Particularly preferred compounds according to the invention
include:
2-cyclopropylamino-4-morpholino-6-n-propyl-1,3,5-triazine,
2-cyclopropyl-4-cyclopropylamino-6-morpholino-1,3,5-triazine
hydrochloride,
2-cyclobutyl-4-cyclopropylamino-6-morpholino-1,3,5-triazine,
2-cyclopropyl-4-cyclopropylamino-6-thiomorpholino-1,3,5-triazine,
and
2-cyclopropyl-4-cyclopropylamino-6-thiomorpholino-1,3,5-triazine
S,S-dioxide.
The present invention also relates to the non-toxic
pharmaceutically acceptable acid addition salts of the substituted
cyclopropylamino-1,3,5-triazines of formula I. As examples of
pharmaceutically acceptable acids there may be mentioned mineral
acids, such as hydrochloric, hydrobromic, sulfuric, nitric and
phosphoric acid and organic acids, such as acetic, citric,
tartaric, benzoic, salicylic and maleic acid.
When the molecule contains one or more asymmetric carbon atoms, the
compounds of formula I may be either in the form of a racemic
mixture or in the form of one of the enantiomers. These various
forms are also within the scope of the present invention.
The substituted cyclopropylamino-1,3,5-triazines according to the
present invention can be prepared by one of the following
processes:
(a) A chloro-cyclopropylamino-1,3,5-triazine of the formula II is
reacted with an amine of the formula R.sub.2 H (III) according to
the equation ##STR3## In these formulae, R.sub.1 and R.sub.2 have
the meanings given above.
(b) A chloro-1,3,5-triazine of the formula IV is reacted with
cyclopropylamine of the formula V according to the equation
##STR4## In these formulae, R.sub.1 and R.sub.2 have the meanings
given above.
(c) An N-cyclopropylbiguanide of the formula VI is reacted with an
alkyl ester of the formula VII by heating under reflux for several
hours in an aliphatic alcohol in the presence of an alkali metal
alcoholate according to the equation ##STR5## In these formulae,
R.sub.1 and R.sub.2 have the meanings given above and Alk
represents an alkyl radical having 1 to 4 carbon atoms, preferably
the ethyl radical.
(d) A cyclopropylamino-1,3,5-triazine of the formula I, wherein
R.sub.1 has the meaning given above and R.sub.2 represents a
thiomorpholino or 3-thiazolidinyl radical, is oxidized in order to
prepare the cyclopropylamino-1,3,5-triazines of the formula I,
wherein R.sub.2 represents a thiomorpholino S-oxide, thiomorpholino
S,S-dioxide, 3-thiazolidinyl S-oxide or 3-thiazolidinyl S,S-dioxide
radical.
Processes (a) and (b) above are carried out by heating at elevated
temperature for several hours in an inert solvent, preferably
dioxane or isopropyl alcohol; in general, they are carried out by
heating at the boiling point of the solvent used and in the
presence of a base. The base, which is used to neutralize the
hydrochloric acid liberated in the course of the reaction, can be
either the amine which itself is used in the reaction or another
organic base (for example triethylamine) or an inorganic base (for
example potassium carbonate).
The starting compounds of the formula II are prepared by
conventional methods, by reacting a 2,4-dichloro-6-R.sub.1
-1,3,5-triazine of the formula VIII with cyclopropylamine of the
formula V according to the equation ##STR6## In these formulae,
R.sub.1 has the meaning given above.
This reaction is generally carried out at a temperature between
-10.degree. C. and room temperature in an inert solvent, such as
chloroform, and in the presence of an inorganic or organic base,
such as, for example, potassium carbonate, to neutralize the
hydrochloric acid liberated during the reaction.
The starting compounds of the formula IV are also prepared by
conventional methods, by reacting a 2,4-dichloro-6-R.sub.1
-1,3,5-triazine of the formula VIII with an amine of the formula
R.sub.2 H (III) according to the equation ##STR7## In these
formulae, R.sub.1 and R.sub.2 have the meanings given above.
This reaction is generally carried out at a temperature between
0.degree. C. and 20.degree. C., in an inert solvent, such as
chloroform, and in the presence of a base, for example potassium
carbonate.
The 2,4-dichloro-6-R.sub.1 -1,3,5-triazines of the formula VIII
used as starting compounds can be prepared by the process of R.
HIRT et al. (Helv. Chim. Acta, 33, (1950), 1365-1369), which
comprises reacting cyanuric chloride with a suitable
organomagnesium compound of the formula R.sub.1 MgX, wherein
R.sub.1 has the meaning given above and X represents a halogen
atom, preferably an iodine or bromine atom.
The starting compounds of the formula VI, used in process (c), are
prepared by a two-step process:
(1) reacting cyclopropylamine of the formula V with the sodium salt
of cyanoguanidine of the formula IX to obtain the
N-cyano-N'-cyclopropylguanidine of the formula X according to the
equation ##STR8##
(2) heating N-cyano-N'-cyclopropylguanidine of the formula X with
an amine of the formula R.sub.2 H (III) at a temperature of about
160.degree. C. for several hours under an inert atmosphere to
obtain the N-cyclopropylbiguanide of the formula VI according to
the equation ##STR9## In these formulae, R.sub.2 has the meaning
given above.
As far as process (d) is concerned, wherein a
cyclopropylamino-1,3,5-triazine of the formula I substituted by a
thiomorpholino or 3-thiazolidinyl radical and prepared according to
one of the processes (a), (b) or (c), is oxidized, this process
results in the formation of the corresponding S-oxide or
S,S-dioxide derivative according to the conditions of operation
used to carry out the oxidation.
This oxidation is generally carried out by means of potassium
peroxomonosulfate (commercialized under the name oxone, 2
KHSO.sub.5.KHSO.sub.4.K.sub.2 SO.sub.4).
The S,S-dioxide derivative is obtained when the reaction is carried
out at a temperature between 10.degree. and 20.degree. C. and in
the presence of 1 to 2 moles of oxone per mole of the compound of
formula I to be oxidized. On the contrary, the S-oxide derivative
is obtained when the reaction temperature is maintained between
-5.degree. C. and +5.degree. C. and only about 0.5 mole of oxone
per mole of the compound of formula I is used.
The non-toxic pharmaceutically acceptable acid addition salts can
be prepared from the substituted cyclopropylamino-1,3,5-triazines
of the formula I by methods which are known per se.
The following examples illustrate the present invention without
limiting it .
EXAMPLE 1
Preparation of the starting 2,4-dichloro-6-R.sub.1 -1,3,5-triazines
of the formula VIII
2,4-Dichloro-6-ethyl-1,3,5,-triazine.
1.5 equivalent of ethylmagnesium bromide dissolved in diethyl ether
(prepared by reacting magnesium with ethyl bromide) are added
dropwise to a suspension of one equivalent of cyanuric chloride in
toluene, while keeping the temperature of the reaction mixture
between 10.degree. and 15.degree. C. After the addition, this
mixture is kept at room temperature for one hour. An aqueous
solution containing 1.5 equivalent of hydrochloric acid is then
added. The two phases are separated, the organic phase is dried
over sodium sulfate, then the solvent is removed under reduced
pressure. 2,4-dichloro-6-ethyl-1,3,5-triazine is purified by
distillation under reduced pressure. Yield: 63%. B.P.: 83.degree.
C./17 mbars.
The compounds summarized in Table I are prepared in the same
manner.
TABLE I ______________________________________
2,4-dichloro-6-R.sub.1 -1,3,5-triazines B.P. .degree.C./ Yield
R.sub.1 Solvent (1) Solvent (2) mbars (in %)
______________________________________ methyl (3) Et.sub.2 O
toluene 80-82/16 40 n-propyl Et.sub.2 O toluene 110/25 60 isopropyl
Et.sub.2 O toluene 87/20 29 cyclopropyl Et.sub.2 O benzene (4) --
cyclobutyl THR toluene (4) -- cyclopentyl Et.sub.2 O benzene 135/13
25 ______________________________________ Et.sub.2 O: diethyl
ether; THF: tetrahydrofuran. (1): solvent used to prepare the
organomagnesium compound, (2): aromatic solvent used for the
dispersion of cyanuric chloride, (3): organomagnesium compound
prepared from methyl iodide, (4): the reaction product is not
isolated by distillation: after addition of the organomagnesium
compound, the reaction mixture is concentrated and the residue is
taken up in anhydrous diethyl ether. The mixture is filtered on
neutral Dicalite, the filtrate is evaporated and the residue is
used as such in the following step.
EXAMLE 2
Preparation of the cyclopropylamino-1,3,5-triazines of the formula
I according to process (a)
A. Preparation of the starting
chloro-cyclopropylamino-1,3,5-triazines of the formula II.
2-Chloro-4-cyclopropylamino-6-methyl-1,3,5-triazine (new
compound).
1 mole of cyclopropylamine dissolved in chloroform is added to a
molar solution of 2,4-dichloro-6-methyl-1,3,5-triazine in
chloroform, previously cooled to -10.degree. C. After the addition,
the mixture is allowed to return to room temperature. The mixture
is then cooled again to about 0.degree. C. and an aqueous solution
containing 1 mole of potassium carbonate is added. Stirring is
continued for 1 to 2 hours at room temperature. The organic phase
is separated off and dried over sodium sulfate, and the solvent is
evaporated under reduced pressure. The residue is recrystallized
from hexane. 2-Chloro-4-cyclopropyl-amino-6-methyl-1,3,5-triazine
is thus obtained. Yield: 75%. M.P.: 117.degree.-119.degree. C.
The following new compounds are prepared in the same manner:
2-Chloro-4-cyclopropylamino-6-ethyl-1,3,5-triazine.
The residue obtained after evaporation of the solvent (crude yield:
100%) is used as such in the following step.
2-Chloro-4-cyclopropylamino-6-n-propyl-1,3,5-triazine.
Yield: 100%. B.P.: 125.degree./0.4 mbar.
2-Chloro-4-cyclopropylamino-6-isopropyl-1,3,5-triazine.
This compound is recrystallized from hexane. Yield: 74%. M.P.:
79.degree.-80.degree. C.
2-Chloro-4-cyclopropyl-6-cyclopropylamino-1,3,5-triazine.
This compound is recrystallized from hexane. Yield (calculated on
the basis of cyanuric chloride): 71.5%. M.P.: 66.degree.-67.degree.
C.
2-Chloro-4-cyclobutyl-6-cyclopropylamino-1,3,5-triazine.
Crude yield (calculated on the basis of the cyanuric chloride):
23%. The product is used as such, without other purification, in
the following step.
2-Chloro-4-cyclopentyl-6-cyclopropylamino-1,3,5-triazine.
Yield (crude): 100%. The crude product is used as such in the
following step.
B. Preparation of the cyclopropylamino-1,3,5-triazines of the
formula I.
1. 2-Cyclopropylamino-4-morpholino-6-n-propyl-1,3,5-triazine
(compound 1).
45 ml (0.45 mole) of morpholine dissolved in 200 ml of dioxane are
added to a solution containing 43 g (0.163 mole) of
2-chloro-4-cyclopropylamino-6-n-propyl-1,3,5-triazine in 300 ml of
dioxane, while maintaining the mixture at room temperature. After
the addition, the mixture is heated under reflux for one to two
hours. The reaction mixture is then allowed to return to room
temperature and the precipitate is filtered off. The filtrate is
concentrated and the residue is redissolved in dichloromethane. The
solution is washed with water. The organic phase is separated off
and dried over sodium sulfate. The solvent is evaporated under
reduced pressure. The residue is purified by chromatography on
silica (eluent: 98.5:1.5 (v/v) dichloromethane-ethanol) and the
product is finally recrystallized from diethyl ether. 36.2 g of
2-cyclopropylamino-4-morpholino-6-n-propyl-1,3,5-triazine are
obtained. Yield: 85%. M.P.: 104.degree. C.
Analysis for C.sub.13 H.sub.21 N.sub.5 O in %: calc.: C 59.29; H
8.04; N 26.59; found: 59.20; 8.15; 26.43.
The compounds summarized in Table II are prepared in the same
manner.
TABLE II
__________________________________________________________________________
##STR10## Analyses Compound R.sub.1 R.sub.2 Yield (%) M.P.
(.degree.C.) calc. % found %
__________________________________________________________________________
2 ethyl morpholino 61 152(1) C 50.44 50.00 H 7.05 7.06 N 24.51
24.10 Cl.sup.- 12.40 12.50 3 cyclopropyl morpholino 67.4 183(1) C
52.44 52.77 H 6.72 6.80 N 23.53 22.16 Cl.sup.- 11.93 11.92 4
cyclobutyl morpholino 80.7 119 C 61.09 61.68 H 7.64 7.67 N 25.82
25.35 5 n-propyl 4-hydroxy- 63.9 102 C 60.65 60.98 piperidino H
8.30 8.46 N 25.27 24.04 6 n-propyl N(CH.sub.2 CH.sub.2 OH).sub.2 68
124(1) C 49.13 49.54 H 7.56 7.48 N 22.05 22.85 Cl.sup.- 11.18 11.18
7 cyclopropyl N(CH.sub.2 CH.sub.2 OH).sub.2 30.8 94 C 55.91 55.90 H
7.53 7.53 N 25.09 24.78 8 cyclopentyl morpholino 40 152(1) C 55.30
55.64 H 7.33 7.44 N 21.50 21.31 Cl.sup.- 10.90 10.84
__________________________________________________________________________
(1) Hydrochloride: prepared by addition of one equivalent of
hydrochloric acid in diethyl ether to one equivalent of the free
base dissolved in diethyl ether. 2.
2Cyclopropylamino-4-methyl-6-(8-oxa-3-azabicyclo[3,2,1]oct-3-yl)-1,3,5-tr
azine hydrochloride (compound 9). 2 equivalents of triethylamine
dissolved in dioxane are added to one equivalent of
8oxa-3-azabicyclo[3,2,1]octane hydrochloride suspended in the same
solvent.
1 equivalent of 2-chloro-4-cyclopropylamino-6-methyl-1,3,5-triazine
dissolved in dioxane is then added. The mixture is heated under
reflux for some hours. It is cooled to room temperature and the
precipitate which has formed is filtered off. The filtrate is
evaporated under reduced pressure and the residue is redissolved in
dichloromethane. The solution is washed with water. The organic
phase is separated off and dried over sodium sulfate, and the
solvent is then evaporated under reduced pressure. The residue thus
obtained is purified by chromatography on silica (eluent: 98:2
(v/v) dichloromethane-ethanol).
2-cyclopropylamino-4-methyl-6-(8-oxa-3-azabicyclo[3,2,1[oct-3-yl)-1,3,5-tr
iazine hydrochloride is prepared by addition of one equivalent of
hydrochloric acid to the free base in diethyl ether. Yield: 63%.
M.P.: 220.degree.-223.degree. C.
Analysis for C.sub.13 H.sub.19 N.sub.5 O.HCl in %: calc. C 52.44; H
6.77; N 23.52; Cl.sup.- 11.92; found 52.40; 6.74; 23.43; 11.84.
The 8-oxa-3-azabicyclo[3,2,1]octane used as the starting compound
in this example is a known compound; it has been prepared according
to the method of F. H. NEWS et al. (J. Chem. Soc., 1948,
115-158).
The compounds summarized in Table III are prepared in the same
manner.
TABLE III
__________________________________________________________________________
##STR11## Analyses Compound R.sub.1 R.sub.2 Yield (%) M.P.
(.degree.C.) calc. % found %
__________________________________________________________________________
10 ethyl OABCO(2) 74 156-159 C 53.93 53.81 (1) H 7.11 7.14 N 22.46
22.22 Cl.sup.- 11.37 11.49 11 n-propyl OABCO 72 129-133 C 55.29
54.97 (1) H 7.42 7.40 N 21.49 20.98 Cl.sup.- 10.88 10.86 12
isopropyl OABCO 59 149-153 C 55.29 55.10 (1) H 7.42 7.39 N 21.49
21.25 Cl.sup.- 10.88 10.92 13 cyclopropyl 3-HO-1-azet- 30.4 111 C
58.30 58.42 idinyl H 6.88 6.84 N 28.34 28.20 14 cyclopropyl
3-CH.sub.3 O-1- 47.3 64 C 59.77 60.05 azetidinyl H 7.28 7.30 N
26.82 26.50 15 n-propyl 3-HO-1-azet- 46.4 76 C 57.83 57.64 idinyl H
7.63 7.66 N 28.11 27.99 16 cyclopropyl OABCO 35 177 C 55.64 55.59 H
6.80 6.84 N 21.64 21.40 17 cyclopropyl 3-oxo-1-azet- 17.1 192-194 C
51.15 50.36 idinyl (1) H 5.68 5.74 N 24.87 24.30
__________________________________________________________________________
(1) Hydrochloride (2) OABCO = 8oxa-3-azabicyclo[3,2,1]oct-3-yl
The 3-azetidinone hydrochloride used as the starting compound for
the synthesis of compound 17 is known; it has been prepared
according to the method of H. BAUMANN et al. (Helv. Chim. Acta, 71,
(1988), 1035).
3. a.
2-Cyclopropyl-4-cyclopropylamino-6-thiomorpholino-1,3,5-triazine
(compound 18).
12.2 g of 2-chloro-4-cyclopropyl-6-cyclopropylamino-1,3,5-triazine
(0.057 mole), 5.97 g of thiomorpholine (0.057 mole) and 8 g of
potassium carbonate (0.057 mole) are mixed in 100 ml of isopropyl
alcohol and the mixture is heated at 75.degree.-80.degree. C. for 2
hours. It is cooled, the salts are filtered off and the filtrate is
evaporated to dryness. The residue is taken up in 200 ml of
dichloromethane, the solution is washed with water and dried over
sodium sulfate and the solvent is evaporated. The product obtained
is crystallized from a 1:2 (v/v) mixture of ethyl acetate-hexane to
give 13.18 g of
2-cyclopropyl-4-cyclopropylamino-6-thiomorpholino-1,3,5-triazine.
Yield: 83.5%. M.P.: 133.degree.-134.degree. C.
Analysis for C.sub.13 H.sub.19 N.sub.5 S in %: calc.: C 56.32; H
6.86; N 25.27; S 11.55; found: 56.06; 6.93; 24.90; 11.40.
The following compounds are prepared in the same manner:
b. 2-Cyclopropyl-4-cyclopropylamino-6-thiomorpholino-1,3,5-triazine
S-oxide (compound 19). Yield: 23%. M.P.: 167.degree.-168.degree.
C.
Analysis for C.sub.13 H.sub.19 N.sub.5 OS in %: calc.: C 53.24; H
6.48; N 23.89; S 10.92; found: 53.10; 6.52; 23.46; 10.62.
c. 2-Cyclopropyl-4-cyclopropylamino-6-thiomorpholino-1,3,5-triazine
S,S-dioxide (compound 20). Yield: 17%. M.P.:
178.degree.-179.degree. C.
Analysis for C.sub.13 H.sub.19 N.sub.5 O.sub.2 S in %: calc.: C
50.49; H 6.15; N 22.65; S 10.36; found: 50.72; 6.18; 22.56;
10.35.
d.
2-Cyclopropyl-4-cyclopropylamino-6-(3-thiazolidinyl)-1,3,5-triazine
(compound 21). Yield: 61.2%. M.P.: 110.degree.-111.degree. C.
Analysis for C.sub.12 H.sub.17 N.sub.5 S in %: calc.: C 54.75; H
6.46; N 26.61; S 12.17; found: 54.83; 6.46; 26.68; 12.30.
e.
2-Cyclopropyl-4-cyclopropylamino-6-(3-thiazolidinyl)-1,3,5-triazine
S,S-dioxide (compound 22). Yield: 35.6%. M.P.:
142.degree.-143.degree. C.
Analysis for C.sub.12 H.sub.17 N.sub.5 O.sub.2 S in %: calc.: C
48.81; H 5.76; N 23.73; S 10.87; found: 49.11; 6.78; 23.69;
10.96.
The thiazolidine 1,1-dioxide hydrochloride used as starting
material for the synthesis of compound 22 above has been prepared
according to a process in 3 steps:
a. N-tert-butoxycarbonyl-thiazolidine.
24 g (0.11 mole) of di-tert-butyl dicarbonate dissolved in 50 ml of
dioxane are added dropwise to a solution of 8.9 g (0.1 mole) of
thiazolidine in 50 ml of dioxane and 50 ml of water, while
maintaining the pH between 10 and 10.5 by addition of soda lye. The
mixture is stirred at room temperature for 6 hours. The formed
salts are filtered off and the organic solvent is evaporated under
reduced pressure. The aqueous residue is extracted with
dichloromethane (2.times.50 ml), the organic phase is decanted,
dried over sodium sulfate and the solvent is evaporated off. The
residue is purified by distillation under reduced pressure. 16.3 g
of N-tert-butoxycarbonyl-thiazolidine are obtained. Yield: 86.2%.
B.P.: 56.degree.-57.degree. C./6.7 mbars.
b. N-tert-butoxycarbonyl-thiazolidine 1,1-dioxide. 30.7 g (0.05
mole) of oxone (2KHSO.sub.5.KHSO.sub.4.K.sub.2 SO.sub.4) dissolved
in 300 ml of water are added dropwise to a solution of 7.3 g
(0.0385 mole) of N-tert-butoxycarbonyl-thiazolidine dissolved in
100 ml of dichloromethane and 200 ml of methanol at room
temperature. The mixture is stirred at 20.degree. C. for 24 hours,
500 ml of water are then added, the mixture is extracted with
dichloromethane (3.times.200 ml), the organic phase is dried over
sodium sulfate and the solvent is evaporated off. The residue
crystallizes from 300 ml of isopropyl ether. 6.58 g of
N-tert-butoxycarbonyl-thiazolidine 1,1-dioxide are obtained. Yield:
74.4%. M.P.: 106.degree.-107.degree. C.
Analysis for C.sub.8 H.sub.15 NO.sub.4 S in %: calc.: C 43.44; H
6.79; N 6.33; S 14.48; found: 43.52; 6.78; 6.33; 14.36.
c. Thiazolidine 1,1-dioxide hydrochloride
1.8 g (0.0081 mole) of N-tert-butoxycarbonyl-thiazolidine
1,1-dioxide and 50 ml of a 2N hydrochloric acid solution in diethyl
ether are mixed. The suspension is stirred at 20.degree. C. for 6
hours, then allowed to stand for 48 hours. The white precipitate of
thiazolidine 1,1-dioxide hydrochloride is filtered off, washed with
diethyl ether and dried. 0.83 g of product is thus obtained. Yield:
65.3%. M.P.: 173.degree.-174.degree. C.
Analysis for C.sub.3 H.sub.7 NO.sub.2 S.HCl in %: calc.: C 22.86; H
5.08; N 8.89; found: 23.29; 5.01; 8.69.
EXAMPLE 3
Preparation of the cyclopropylamino-1,3,5-triazines of the formula
I according to process (b)
A. Preparation of the starting chloro-1,3,5-triazines of the
formula IV.
2-Chloro-4-cyclopropyl-6-morpholino-1,3,5-triazine.
A solution of 2.61 g (0.03 mole) of morpholine in 20 ml of
chloroform is added in the course of 30 minutes to a solution of
5.7 g (0.03 mole) of 2,4-dichloro-6-cyclopropyl-1,3,5-triazine in
50 ml of chloroform, cooled to between 3.degree. and 5.degree. C.
The temperature of the mixture is allowed to return to about
10.degree. C., then the mixture is cooled again to 5.degree. C. and
a solution of 4.14 g (0.03 mole) of potassium carbonate in 15 ml of
water is added dropwise. The mixture is then stirred at room
temperature for two hours. 30 ml of water are added and the organic
phase is separated off. The solution is washed with water and dried
over sodium sulfate, and the solvent is evaporated under reduced
pressure. The residue is purified by chromatography on silica
(eluent: 96.5:3.5 (v/v) dichloromethane-ethylacetate) and then
recrystallized from hexane. 5.5 g of
2-chloro-4-cyclopropyl-6-morpholino-1,3,5-triazine are thus
obtained. Yield: 76.2%. M.P.: 99.degree.-100.degree. C.
Analysis for C.sub.10 H.sub.13 ClN.sub.4 O in %: calc.: C 49.89; H
5.41; N 23.28; Cl 14.76; found: 49.91; 5.44; 23.06; 14.46;
B. Preparation of the cyclopropylamino-1,3,5-triazines of the
formula I.
2-Cyclopropyl-4-cyclopropylamino-6-morpholino-1,3,5-triazine.
2.85 g (0.050 mole) of cyclopropylamine dissolved in 20 ml of
dioxane are added to a solution of 5.1 g (0.021 mole) of
2-chloro-4-cyclopropyl-6-morpholino-1,3,5-triazine in 50 ml of
dioxane at room temperature. The mixture is heated under reflux for
5 hours. The solvent is then evaporated under reduced pressure and
the residue is dissolved in 50 ml of dichloromethane and 50 ml of
water. The organic phase is separated off and dried over sodium
sulfate and the solvent is evaporated under reduced pressure. The
residue crystallizes from cold hexane. 5.22 g of product are
obtained. This product forms a hydrochloride in an ethanol-diethyl
ether mixture. 5.1 g of
2-cyclopropyl-4-cyclopropylamino-6-morpholino-1,3,5-triazine
hydrochloride, which is identical to compound 3 prepared in example
2. B.1., are obtained. Yield: 81.7%. M.P.: 183.degree.-184.degree.
C. (acetonitrile).
Analysis for C.sub.13 H.sub.19 N.sub.5 O.HCl in %: calc.: C 52.44;
H 6.72; N 23.53; Cl.sup.- 11.93; found: 52.46; 6.73; 23.44;
11.89.
EXAMPLE 4
Preparation of the cyclopropylamino-1,3,5-triazines of the formula
I according to process (c)
A. N-cyano-N'-cyclopropylguanidine.
9.36 g (0.1 mole) of cyclopropylamine hydrochloride and 8.9 g (0.1
mole) of the sodium salt of cyanoguanidine are suspended in 100 ml
of n-butanol and 8 ml of water. The mixture is heated under reflux
for 2 hours. The suspension is filtered and the filter cake is
washed with 100 ml of n-butanol. The filtrate is evaporated under
reduced pressure. The residual oil is taken up in 300 ml of
acetonitrile. The mixture is heated to reflux, filtered hot and the
filter cake is washed with acetonitrile. The filtrate is evaporated
off and the oil obtained crystallizes slowly. 7.7 g of
N-cyano-N'-cyclopropylguanidine are obtained. Yield: 62%. M.P.:
106.degree. C. (isopropyl alcohol/diethyl ether).
Analysis for C.sub.5 H.sub.8 N.sub.4 in %: calc.: C 48.37; H 6.49;
N 45.13; found: 48.40; 6.50; 45.15.
B. Preparation of the starting N-cyclopropylbiguanides of the
formula VI.
1. N-[imino(cyclopropylamino)methyl]-4-morpholinecarboximidamide
hydrochloride.
A mixture of 4.0 g (32.3 mmoles) of N-cyano-N'-cyclopropylguanidine
and 3.98 g (32.3 mmoles) of morpholine hydrochloride is heated at
160.degree. C. under a nitrogen atmosphere for 2 and a half hours.
The solid mass obtained is dissolved in 200 ml of boiling isopropyl
alcohol. The mixture is filtered hot and the filtrate is
concentrated until a solid suspension appears. The mixture is then
cooled to room temperature and 200 ml of diethyl ether are added.
The reaction product crystallizes. It is filtered, washed with
diethyl ether and dried. 6.8 g of
N-]imino(cyclopropylamino)methyl]-4-morpholinecarboximidamide
hydrochloride are obtained. Yield: 85%. M.P.:
195.degree.-196.degree. C.
Analysis for C.sub.9 H.sub.17 N.sub.5 O.HCl in %: calc.: C 43.63; H
7.32; N 28.27; Cl.sup.- 14.21; found: 43.60; 7.35; 27.93;
14.18.
2.
N-[imino(cyclopropylamino)methyl]-4-thiomorpholinecarboximidamide
S,S-dioxide hydrochloride.
This compound is prepared in the same manner as the previous
compound by reacting thiomorpholine 1,1-dioxide hydrochloride
(British patent No. 874,519) with N-cyano-N'-cyclopropylguanidine
at 160.degree. C. for 6 hours. Yield: 58.4%.
The product obtained, which is 90% pure (chromatography), is used
as such in the following reaction.
C. Preparation of the cyclopropylamino-1,3,5-triazines of the
formula I.
1.
2-Cyclopropylamino-4-(1-methylcylopropyl)-6-morpholino-1,3,5-triazine
(compound 24).
0.48 g (20 mmoles) of sodium is dissolved in 20 ml of anhydrous
ethanol. This solution is added to an ethanol solution containing
2.48 g (10 mmoles) of
N-[imino(cyclopropylamino)methyl]-4-morpholinecarboximidamide
hydrochloride, and 2.82 g (22 mmoles) of ethyl
1-methylcyclopropane-carboxylate are then also added to the
mixture. The mixture is then heated under reflux under nitrogen for
88 hours. It is cooled, the alcohol is evaporated under reduced
pressure and the residue is redissolved in 50 ml of water and 200
ml of dichloromethane. The aqueous phase is separated off and the
organic phase is washed twice with 50 ml of water and dried over
magnesium sulfate. The solvent is evaporated under reduced pressure
and the residue is recrystallized from a 1:1 (v/v) mixture of
petroleum ether-dichloromethane. 0.49 g of
2-cyclopropylamino-4-(1-methylcyclopropyl)-6-morpholino-1,3,5-triazine
is obtained. Yield: 17%. M.P.: 94.degree.-95.degree.% C.
Analysis for C.sub.14 H.sub.21 N.sub.5 O in %: calc.: C 61.06; H
7.69; N 25.44; found: 61.15; 7.66; 25.56.
The following compounds are prepared in the same manner:
2.
2-Cyclopropylamino-4-(2-methylcyclopropyl)-6-morpholino-1,3,5-triazine
hydrochloride (compound 25).
Heating period: 62 hours.
Yield: 38.7%. M.P.: 177.degree.-178.degree. C.
Analysis for C.sub.14 H.sub.21 N.sub.5 O.HCl in %: calc.: C 53.93;
H 7.06; N 22.47; Cl.sup.- 11.40; found: 54.04; 7.14; 22.19;
11.27.
3. 2-Cyclobutyl-4-cyclopropylamino-6-thiomorpholino-1,3,5-triazine
S,S-dioxide hydrochloride (compound 26).
Heating period: 18 hours.
Yield: 39%. M.P.: 220.degree.-225.degree. C. (dec.).
Analysis for C.sub.14 H.sub.21 N.sub.5 O.sub.2 S.HCl in %: calc.: C
46.73; H 6.12; N 19.47; S 9.87; Cl.sup.- 8.90; found: 46.24; 6.02;
19.14; 9.78; 8.70.
4.
2-Cyclopropylamino-4-(2-methylcyclopropyl)-6-thiomorpholino-1,3,5-triazine
S,S-dioxide (compound 27).
Heating period: 27 hours.
Yield: 93.9%. M.P.: 180.degree.-182.degree. C.
Analysis for C.sub.14 H.sub.21 N.sub.5 O.sub.2 S in %: calc.: C
52.01; H 6.50; N 21.67; S 9.91; found: 52.00; 6.54; 21.36;
9.89.
5.
2-Cyclopropylamino-4-(2,2-dimethylcyclopropyl)-6-thiomorpholino-1,3,5-tria
zine S,S-dioxide (compound 28).
Heating period: 6 days.
Yield: 40.1%. M.P.: 170.degree.-172.degree. C.
Analysis for C.sub.15 H.sub.23 N.sub.5 O.sub.2 S in %: calc.: C
53.41; H 6.82; N 20.77; S 9.50; found: 54.02; 6.86; 20.88;
9.39.
6.
2-Cyclopropylamino-4-(2-n-propylcyclopropyl)-6-thiomorpholino-1,3,5-triazi
ne S,S-dioxide (compound 29).
Heating period: 20 hours in an autoclave at 110.degree. C.
Yield: 28.7%. M.P.: 148.degree.-149.degree. C.
Analysis for C.sub.16 H.sub.25 N.sub.5 O.sub.2 S in %: calc.: C
54.70; H 7.12; N 19.84; found: 54.71; 7.10; 20.05.
Example 5
Preparation of the cyclopropylamino-1,3,5-triazines of the formula
I according to process (d)
A.
2-Cyclopropyl-4-cyclopropylamino-6-(3-thiazolidinyl)-1,3,5-triazine
S,S-dioxide (compound 22).
A solution of 30.7 g (0.05 mole) of oxone
(2KHSO.sub.5.KHSO.sub.4.K.sub.2 SO.sub.4) in 75 ml of water is
added dropwise to a solution of 6.58 g (0.025 mole) of
2-cyclopropyl-4-cyclopropylamino-6-(3-thiazolidinyl)-1,3,5-triazine
(compound 21) in 75 ml of dichloromethane and 150 ml of methanol,
at a temperature between 10.degree. and 15.degree. C. The mixture
is stirred at room temperature for 3 hours. 3 g of oxone dissolved
in 10 ml of water are then added again and stirring is continued
for 2 hours. 200 ml of water are added, the mixture is extracted
with dichloromethane (3.times.100 ml), the organic phase is
separated off and dried over sodium sulfate and the solvent is
evaporated. The residue is purified by chromatography on silica
(eluent: 98:2 (v/v) dichloromethane-ethanol).
3 g of
2-cyclopropyl-4-cyclopropylamino-6-(3-thiazolidinyl)-1,3,5-triazine
S,S-dioxide identical to the compound prepared in example 2.B.3.e.
are obtained.
Yield: 40.7%. M.P.: 142.degree.-143.degree. C. (ethyl
acetate-hexane).
B.
2-Cyclopropyl-4-cyclopropylamino-6-(3-thiazolidinyl)-1,3,5-triazine
S-oxide (compound 23).
A solution of 15.35 g (0.025 mole) of oxone in 150 ml of water is
added dropwise and without exceeding the temperature of 5.degree.
C., to a solution of 11.85 g (0.045 mole) of
2-cyclopropyl-4-cyclopropylamino-6-(3-thiazolidinyl)-1,3,5-triazine
(compound 21) in 150 ml of dichloromethane and 300 ml of methanol,
cooled to about 0.degree. C. The mixture is then further stirred at
5.degree. C. for 30 minutes. The precipitate of mineral salts is
filtered off on Hyflo-cel. The organic phase is eliminated by
decantation. The aqueous phase is diluted with 400 ml of water and
extracted with dichloromethane (2.times.200 ml). The organic phase
is dried over sodium sulfate and the solvent evaporated off. The
evaporation residue is taken up in 100 ml of dichloromethane,
filtered off and concentrated. A first crop of 2.83 g of a solid
product is obtained. The aqueous phase is rendered alkaline and
extracted with dichloromethane (3.times.100 ml), the organic phase
is dried over sodium sulfate and the solvent is evaporated. 8.41 g
of a solid residue are thus obtained (second crop). The two crops
are brought together and purified by chromatography on silica
(eluent: 93:3 (v/v) dichloromethane-ethanol). 7.66 g of
2-cyclopropyl-4-cyclopropylamino-6-(3-thiazolidinyl)-1,3,5-triazine
S-oxide, crystallized from a 1:3 (v,v) mixture of ethyl
acetate-hexane, are obtained.
Yield: 61%. M.P.: 136.degree.-137.degree. C.
Analysis for C.sub.12 H.sub.17 N.sub.5 OS in %: calc.: C 51.61; H
6.09; N 25.03; S 11.47; found: 51.79; 6.10; 25.09; 11.50.
As indicated above, the substituted
cyclopropylamino-1,3,5-triazines of the formula I and their
non-toxic pharmaceutically acceptable acid addition salts have the
property of correcting the effects of hypofunctioning of the
cholinergic system and consequently have a favorable activity on
mnemic processes. In addition, they have a central serotonergic and
a bronchospasmolytic activity; they oppose the release of mastocyte
mediators, have an anti-inflammatory and anti-edema activity and
potentiate the effect of a .beta.-adrenergic agonist on muscle
relaxation.
The pharmacological tests described below demonstrate these various
advantageous properties.
1. Activity on mnemic processes
The compounds according to the present invention were studied with
the aim of demonstrating on the one hand their property of
promoting learning, expressed as the reduction in the number of
trials needed to achieve a predetermined criterion, and on the
other hand their property of counteracting the amnesia caused by
administration of scopolamine.
To this end, the method of multiple-trial passive avoidance has
been used. This method is well-known for evaluating the effects
which a product exerts on the memory and learning (A. FINE et al.,
Proc. Natl. Acad. Sci. USA, 82, (1985), 5227-5230).
The test is carried out on male Sprague-Dawley rats (160-200 g),
which are kept in standard cages throughout the experiment. The
apparatus used is a transparent square cage with 35 cm sides and 25
cm high, fitted with a grid floor which can be electrified. A
rubber insulating mat (10.times.17 cm) is placed on the floor in
one of the corners of the cage.
To evaluate whether a compound can promote learning, the following
test is carried out.
Each animal is placed on the rubber mat and the time the animal
takes to decide to leave this position to explore the cage is
recorded. After 20 seconds of exploration, the animal receives an
electric shock (3 seconds duration) in the paws, causing an escape
reaction. The rat is immediately removed from the apparatus and
replaced in its original cage. This experiment is repeated until
the animal remains on the rubber mat for at least 180 seconds in
order to avoid the electric shock. The learning is expressed by the
average number of trials needed to reach a period of 180 seconds
remaining on the mat. A period of remaining on the rubber mat of
180 seconds is regarded as being the maximum performance to be
realized by the animal to avoid the electric shock. Rats which
remain on the mat for this period have acquired the avoidance
reflex and are replaced in their original cage without receiving
the electric shock.
To evaluate whether a compound is capable of promoting mnemic
retention in the course of time, the following experiment is
carried out. Each animal is subjected to four tests at times 0, 4,
24 and 28 hours. In the first test (time 0), the animal is placed
on the rubber mat and the time which it takes to decide to leave
this position to explore the cage is recorded. After 20 seconds of
exploration, the rat receives an electric shock (3 seconds
duration) in the paws, causing an escape reaction. The rat is
immediately removed from the apparatus and replaced in its original
cage. In the course of the three subsequent tests (times: 4, 24 and
28 hours), the animal is replaced on the rubber mat and the time
taken to leave this position is recorded. As soon as the four paws
of the animal rest on the grid, it receives an electric shock and
is removed immediately from the apparatus.
At the start of the experiment, the rats are divided into 4
homogeneous groups of 15 animals. Thirty minutes before each test,
each group of animals is subjected to a predetermined
treatment:
group 1 receives an intraperitoneal injection of physiological salt
solution;
group 2 receives an intraperitoneal injection of the compound to be
tested;
group 3 receives an intraperitoneal injection of 0.5 mg of
scopolamine and
group 4 receives an intraperitoneal injection of 0.5 mg of
scopolamine and, simultaneously, an intraperitoneal injection of
the compound to be tested.
Groups 1 and 2 are used in the first experiment (learning) and
groups 3 and 4 in the second experiment (mnemic retention).
The results obtained in this test with the compounds according to
the invention are summarized in Table IV. This Table shows the
compound subjected to the test (column 1) and the dose administered
intraperitoneally, expressed in mg/kg (column 2).
The results obtained in the test used to evaluate learning are
given in columns 3 and 4. The figures indicate the average number
of trials needed for a control animal (group 1) or an animal
treated with the compound (group 2) to learn to remain on the
rubber mat for 180 seconds in order to avoid the electric shock.
The results were analyzed by the Student test.
The results obtained in the experiment used to evaluate mnemic
retention are given in columns 5 to 12. In columns 5 to 8, the
figures represent the average periods of remaining on the mat
observed respectively at times 0, 4, 24 and 28 hours for the
animals of the group 3, treated only with scopolamine, and in
columns 9 to 12 the corresponding figures are found for the animals
of group 4, treated simultaneously with scopolamine and with the
compound tested (at the dose shown in the second column).
The favorable influence of a compound in counteracting amnesia
induced by scopolamine is demonstrated by the increase in the
period of remaining on the mat at each observation. The differences
found are analyzed statistically by the Mann-Whitney method.
TABLE IV
__________________________________________________________________________
Mnemic Retention Learning Period of remaining on the mat (in
seconds) Dose average number of trials group 3 group 4 Compound
(mg/kg) group 1 group 2 0 4 24 28 0 4 24 28
__________________________________________________________________________
1 3.0 3.7 1.4 3.7 18.1 19.7 34.3 19.2 42.8 77.2 114.6 2 1.0 3.2 2.7
5.3 7.3 11.2 14.5 7.9 18.9 24.3 36.5 3 1.0 3.0 1.9 5.6 6.7 20.7
17.5 6.5 35.5 105.0 121.0 4 2.8 3.9 1.9 4.5 13.1 20.9 61.3 12.3
44.5 91.5 106.6 5 2.8 3.3 2.3 5.1 7.9 8.6 21.0 14.5 34.2 35.9 73.3
6 3.2 3.4 2.5 3.1 5.5 6.4 12.1 5.3 11.7 24.8 28.7 7 2.8 2.0 1.9 3.7
18.5 38.5 59.6 9.8 34.5 94.0 133.0 9 3.0 2.8 2.9 5.2 4.7 25.4 69.6
3.6 17.3 38.1 65.2 10 3.1 2.7 1.4 2.7 24.1 27.6 52.3 5.7 20.3 78.9
142.0 11 3.3 3.1 1.8 4.2 7.3 5.3 10.3 10.7 21.4 58.6 93.2 12 1.0
2.6 1.7 3.2 20.9 40.6 41.1 3.6 18.0 51.1 94.5 13 2.5 2.3 2.1 5.0
8.9 19.7 34.9 7.7 55.3 92.5 99.0 14 2.6 2.3 1.5 5.3 7.1 21.3 67.7
11.7 36.3 73.9 139.1 15 2.5 2.3 1.9 5.1 12.1 22.7 59.7 9.8 19.5
94.3 112.7 16 3.25 2.5 2.2 6.2 10.3 29.1 34.3 11.3 35.1 118.5 101.1
physostigmine 0.4 2.6 2.3 1.7 1.4 6.9 21.3 1.9 17.8 56.2 53.4
__________________________________________________________________________
From this Table, it can be seen that:
the compounds according to the invention promote learning of the
avoidance reflex: the average number of trials needed to reach the
predetermined criterion (maximum period of remaining on the mat of
180 seconds) is lower for the treated animals (column 4) than for
the control animals (column 3);
the amnesic effect of scopolamine is very pronounced: it can be
seen that the period of remaining on the mat for the animals of
group 3 (columns 5 to 8) are clearly less than the 180 seconds
realized by the controls after an average number of trials (column
3); and, under these conditions, the favorable influence of the
compounds according to the invention in counteracting the amnesic
effect of scopolamine is very clear: the animals of group 4,
treated simultaneously with scopolamine and with a compound
according to the invention, have periods of remaining on the mat,
at each observation, which are considerably longer than those for
the animals of group 3, treated with scopolamine alone (compare the
results of column 5 with those of column 9, 6 with 10, and so
on).
physostigmine exerts a favorable action against the amnesic effect
of scopolamine, similar to that of the compounds according to the
invention, but at a dose which has side effects and is very close
to the toxic dose, which is not the case for the compounds
according to the invention.
2. Serotonergic activity
A decrease in serotonergic activity has been correlated with the
occurrence of affective disorders, such as depression and anxiety.
Injection of rats with serotonin or 5-hydroxytryptophan (5-HTP), a
serotonin agonist, induces paroxysmal shakes of the head, neck and
trunk in this animal, similar to the shuddering of a wet dog
shaking itself. This behavior is called "wet dog shake" (WDS) and
is used as a model to demonstrate the aminergic and in particular
serotonergic properties found in antidepressants (P. BEDARD and C.
J. PYCOCK, Neuropharmacology, 16, (1977), 663-670).
The test is carried out in the morning on male Sprague-Dawley rats
(.+-.180 g), divided the previous day into groups of 8 animals in
residence cages. The cage used for the tests is a transparent
enclosure of 12.times.24.times.30 cm in height, the floor of which
is covered with a layer of sand.
The compounds to be tested, dissolved either in physiological salt
solution or in a citrate buffer of pH 5, are administered
intraperitoneally in a different dose for each group treated. The
control groups receive an intraperitoneal injection of the same
vehicle (either physiological salt solution or citrate buffer).
After administration of the compound, the animals are immediately
placed in the test cage in groups of four at a time, and after a
habituation period of 10 minutes, the number of shakes (WDS) which
occur over a period of 30 minutes is counted.
The mean values of the results are calculated and analyzed
statistically by the Mann-Whitney method.
The mean values of the number of shakes obtained for the compounds
according to the invention administered intraperitoneally at the
doses indicated (in mg/kg) are given in Table V below.
TABLE V ______________________________________ "Wet dog shake"
behavior Dose Average number Compound (mg/kg) of shakes
______________________________________ 1 1.0 7.8 .+-. 1.6 2 1.0
10.0 .+-. 3.5 3 0.3 5.1 .+-. 1.0 1.0 11.9 .+-. 2.7 4 2.8 10.6 .+-.
4.3 5 2.8 8.4 .+-. 1.6 6 1.0 1.9 .+-. 0.5 3.2 3.6 .+-. 1.1 7 2.8
7.9 .+-. 1.8 10 3.1 16.3 .+-. 3.8 11 1.1 4.3 .+-. 1.4 12 3.2 16.3
.+-. 4.2 13 2.5 5.6 .+-. 1.8 14 0.8 2.1 .+-. 0.7 16 1.0 10.6 .+-.
1.8 17 2.8 10.5 .+-. 2.9 18 0.88 10.6 .+-. 3.0 19 0.94 16.0 .+-.
4.7 20 0.99 13.6 .+-. 2.3 21 2.63 5.5 .+-. 2.0 22 0.95 8.1 .+-. 1.7
23 0.89 10.1 .+-. 3.0 24 2.75 6.1 .+-. 2.3 25 1.0 19.6 .+-. 5.0 26
1.15 11.8 .+-. 4.3 5-HTP/carbidopa (1) 100.0 4.7 .+-. 1.5 200.0
19.6 .+-. 3.0 ______________________________________ (1) The
animals are pretreated with the peripheral decarboxylase inhibito
.alpha.-methyldopahydrazine or carbidopa (25 mg/kg, i.p., 30
minutes before the 5HTP); the measurements are made 90 to 120
minutes after the intraperitoneal injection of 5HTP.
(1) The animals are pretreated with the peripheral decarboxylase
inhibitor .alpha.-methyldopahydrazine or carbidopa (25 mg/kg, i.p.,
30 minutes before the 5-HTP); the measurements are made 90 to 120
minutes after the intraperitoneal injection of 5-HTP.
This Table shows that the compounds according to the invention
induce a "wet dog shake" behavior in rats comparable to that caused
by injections of a serotonergic agonist such as 5-HTP in the
presence of carbidopa, but at doses which are clearly lower.
3. Bronchospasmolytic activity
This activity was measured in the Dunkin-Hartley guinea-pig by the
method of H. KONZETT and R. ROESSLER (Naunyn Schmiedebers Arch.
exp. Path. Pharmacol., 195, (1940), 71-74) and compared with that
of theophylline.
The anesthetized (urethane) and curarized (gallamine) guinea-pig is
placed under assisted respiration. The endotracheal pressure is
recorded. Repeated bronchial spasms are induced by successive
intravenous injections (every 5 minutes) of serotonin, histamine or
acetylcholine, respectively, at a dose which is capable of inducing
an increase of the endotracheal pressure of 20 to 50 cm water. The
compound to be tested is also administered intravenously two
minutes before the adminstration of the spasmogen and then in 3 to
4 cumulative doses in increasing amounts at intervals of 15
minutes. Six animals per compound to be tested and per spasmogen
are used. Table VI below shows the doses of the compounds (ID50 in
.mu.mole/kg) which inhibit by 50%, on average over all the animals,
the bronchospasms induced.
TABLE VI ______________________________________ Bronchospasmolytic
Activity Doses (ID50 in .mu.mole/kg) Compound Serotonin Histamine
Acetylcholine ______________________________________ 1 0.01 0.01
0.1 2 0.5 0.3 0.5 3 0.1 0.03 0.07 4 0.03 0.004 0.03 5 0.1 0.06 0.1
6 0.1 0.1 0.3 7 0.4 0.2 0.7 8 0.3 0.1 0.2 9 1.0 0.4 2.1 10 0.3 0.1
0.5 11 0.1 0.1 0.09 12 0.06 0.04 0.2 13 0.6 0.1 1.3 14 0.2 0.09 0.5
15 0.2 0.08 0.3 16 0.009 0.009 0.05 17 0.2 0.1 0.2 18 -- 0.003 --
19 0.04 0.02 0.1 20 0.4 0.03 0.1 24 -- 0.04 -- 26 -- 0.03 --
theophylline 4.6 5.6 10.0
______________________________________
It can be seen from this Table that the compounds according to the
invention have a remarkable bronchospasmolytic activity with
respect to bronchospasms induced by, respectively, serotonin,
histamine or acetylcholine.
4. Anti-inflammatory and anti-edema activity
The reaction between a soluble antigen and antibodies in the
organism may lead to an acute inflammatory reaction accompanied by
release of histamine, modification of vascular permeability and
formation of a localized edema.
The purpose of the "reverse passive Arthus" (RPA) test is to
demonstrate the anti-inflammatory properties of a compound on the
plantar edema induced experimentally by immune complexes in the
Sprague-Dawley rat (P. J. BAILEY and A. STURM, Biochem. Pharmacol.,
32, (1983), 475). To this end, the Arthus reaction is induced by
subplantar administration of 0.1 ml of heterologous antiovalbumin
reaginic serum into the right hind paw and by simultaneous
intravenous injection of 1 ml/kg ovalbumin (25 mg/ml).
The compound to be tested is administered intravenously 30 seconds
before induction of the Arthus reaction, in a least 3 different
doses. A group of 6 rats is used per dose of compound to be tested.
The volume of the paw is measured by plethysmometry before the
Arthus reaction and 3 to 5 hours after induction of the Arthus
reaction, both in the control animals and in the treated animals.
The effect of a compound on the reduction in the edema for each
dose and at each measurement time (3 hours and 5 hours) is
expressed in % of the edema observed in the control animals.
The doses of the compounds (ID30 in .mu.mole/kg) which inhibit by
30%, on average over all the animals, the volume of the edema
observed in the control animals are given in Table VII below.
TABLE VII ______________________________________ Anti-inflammatory
and anti-edema activity Dose (ID30 in .mu.mole/kg) Compound 3 hours
5 hours ______________________________________ 1 2.0 2.0 2 2.3 0.7
3 0.8 0.4 4 9.0 10.0 5 3.1 27.0 6 12.0 9.0 7 7.7 11.0 8 4.0 4.0 9
3.4 4.4 11 0.5 0.4 12 2.5 3.0 13 9.0 4.0 14 16.0 12.0 15 39.0 4.0
16 1.5 2.0 17 1.0 1.0 18 1.0 1.0 19 0.02 0.02 20 1.0 0.5
theophylline 18.0 10.0 ______________________________________
This Table shows that the compounds according to the invention have
an anti-inflammatory and anti-edema activity which is clearly
superior to that of theophylline.
5. Inhibition of the anaphylactic release of histamine
The test has two purposes: on the one hand to evaluate the "in
vitro" inhibitory activity of a compound on the anaphylactic
release of histamine caused by degranulation of mastocytes in the
guinea-pig lungs, and on the other hand to demonstrate a possible
potentiation effect (synergism) on the inhibitory activity of a
.beta.-adrenergic agonist, for example isoprenaline, on this same
release of histamine (E.S.K. ASSEM and H.O. SCHILD, Int. Arch.
Allergy, 40, (1971), 576-589).
Dunkin-Hartley guinea-pigs which are first passively sensitized by
an intravenous injection of 1 ml of isologous antiovalbumin serum
are used.
Twenty-four hours after the injection, the lungs are then perfused
with a Tyrode buffer to evacuate the blood, and then removed and
cut into sections of 1 mm. These sections are divided up into test
tubes (3 sections per tube) to give 10 experimental groups of lung
sections per guinea-pig.
A "positive" control group of lung sections is stimulated by
addition of an ovalbumin solution (0.1 mg/ml) to induce the
anaphylactic release of histamine and serves to determine the
maximum amount of histamine (100%) which can be released.
A "negative" control group which is not stimulated by ovalbumin
serves to determine the naturally released amount of histamine
(spontaneous release).
In three other groups, the lung sections are incubated at
37.degree. C. for 30 minutes in the ovalbumin solution in the
presence of three different doses of the compound to be tested (one
dose per group). To detect a potentiation effect of a compound
according to the invention on the inhibition of histamine release
by isoprenaline, three other groups of lung sections are treated in
the same manner as above, but the incubation medium also contains
isoprenaline at a dose of 10.sup.-7 mole per liter. At this dose,
isoprenaline inhibits the release of histamine by 30 to 40%, which
is verified on a control group treated only with isoprenaline alone
at this dose. In addition, a supplementary control group which is
not stimulated by ovalbumin is used to detect a possible
spontaneous release of histamine by the highest dose of the
compound tested.
In each group, the histamine released in the incubation medium is
measured by spectrofluorometry (D. P. EVANS et al., Life Sci., 12,
(1973), 327-336).
The results obtained enable a minimum inhibitory dose (MID) (proper
effect) to be determined for each compound, that is to say the dose
of the compound at which the amount of histamine released is lower
than that of the "positive" control group and at which this
difference is statistically significant, and a minimum potentiating
dose (MPD), which is the dose of compound which induces an
inhibition greater than that of isoprenaline at a dose of 10.sup.-7
mole per liter.
The minimum inhibitory doses (MID) and the minimum potentiating
doses (MPD), expressed in .mu.mole/l, obtained in this test with
the compounds according to the invention and with theophylline as
the reference substance are given in Table VIII below.
TABLE VIII ______________________________________ Inhibition of the
anaphylactic release of histamine Minimum inhibitory Minimum
potentiating dose dose Compound (in .mu.mole/l) (in .mu.mole/l)
______________________________________ 1 0.1 0.32 2 1.0 3.2 3 0.032
0.32 4 0.32 0.1 5 0.32 3.2 6 1.0 1.0 7 0.32 1.0 8 1.0 10.0 9 3.2
3.2 10 3.2 0.32 11 1.0 0.32 12 0.1 0.1 13 1.0 1.0 14 1.0 0.32 15
10.0 3.2 16 0.1 0.032 17 3.2 1.0 18 0.1 1.0 19 1.0 0.1 20 1.0 0.32
21 0.1 0.32 22 0.1 0.032 24 0.032 0.32 25 0.032 0.032 26 1 0.32
theophylline 100.0 1000.0
______________________________________
It can be seen from this Table that the compounds according to the
invention are much more active than theophylline in inhibiting the
anaphylactic release of histamine (proper effect) and at the same
time have, at low doses, a potentiating effect on the inhibiting
effect of isoprenaline obtained at a dose of 10.sup.-7
mole/liter.
6. Potentiation of the myorelaxing effect of isoprenaline on the
ileum of the guinea-pig
(cf. R. A. TURNER, "Screening Methods in Pharmacology", Ed. Acad.
Press, San Diego, 1965, chapter IV, pages 43-47).
Fragments of the longitudinal muscles of the ileum of
Dunkin-Hartley guinea-pigs (250 to 500 g) attached to an isometric
force indicator are dipped in a Tyrode solution
(37.degree..+-.1.degree. C., pH 7.6; 5.6 mmoles/l of glucose),
oxygenated by passing a stream of gas (mixture of 95% O.sub.2 -5%
CO.sub.2) and tightened with a force of 1 g.
After stabilization of the tension, successive cycles of histamine
injection (histamine dihydrochloride at a dose of 3.2 .mu.moles/l)
are effected by means of a perfusion pump of the Braun type. Each
injection cycle comprises the 6 following successive phases: a rest
phase (duration: 25 seconds), a histamine injection phase
(duration: 6 seconds), a period of muscular contraction (duration:
24 seconds), a first wash with water (duration: 25 seconds), a
period of stabilization, during which the muscle returns to the
starting tension (duration: 35 seconds), followed by a second wash
with water (duration: 25 seconds).
Inhibition cycle: When the successive contractions induced by the
histamine injections have become reproducible, the compound to be
tested is injected immediately after the second wash. The
contraction induced by the following injection of histamine is
measured and compared with the average of the three preceding
contractions caused by the repeated injections of histamine
(control contractions). From the result obtained, the percentage
inhibition of the contraction is calculated. Several cycles of
histamine injection are then performed again, until the
contractions of the muscle become reproducible again; the muscle is
then ready for testing of another compound.
The potentiating effect of a compound is measured in the course of
a so-called "potentiation" cycle, which comprises three successive
inhibition cycles. In the first inhibition cycle, the percentage
inhibition of the contraction obtained by injection of isoprenaline
alone at a dose of 10.sup.-7 mole/l is determined. At this dose,
the percentage inhibition of the contraction is usually between 10
and 25%. In the course of the second inhibition cycle, the
percentage inhibition of the contraction obtained with the compound
to be tested, injected alone at a given dose, is determined. In the
third inhibition cycle, isoprenaline and the compound to be tested
are injected simultaneously, and the percentage inhibition of the
contraction is calculated for the dose used of the compound
tested.
The same experiments are repeated for each of the doses of the
compounds tested.
From the results obtained, the minimum potentiating dose (MPD) is
determined for each compound tested; this dose corresponds to the
dose of compound at which the inhibition obtained with the mixture
of compound and isoprenaline is significantly superior (p<0.05)
to the sum of each of the inhibitions obtained when the compound
and isoprenaline are injected alone.
In Table IX below, the minimum dose, expressed in .mu.mole/l, which
potentiates the myorelaxing effect of isoprenaline administered at
a dose of 10.sup.-7 mole/l, is given for the compounds according to
the invention in comparison with theophylline.
TABLE IX ______________________________________ Potentiation of the
myorelaxing effect of isoprenaline on the ileum of the guinea-pig
Minimum potentiating dose Compound (in .mu.mole/l)
______________________________________ 1 0.032 2 1.0 3 0.032 4
0.032 5 1.0 6 1.0 7 0.32 9 10.0 10 1.0 11 0.032 12 0.01 13 1.0 14
1.0 15 3.2 16 0.032 17 0.32 18 0.1 19 0.32 20 0.1 theophylline
1000.0 ______________________________________
This Table shows that the compounds according to the invention are
much more active than theophylline in potentiating the myorelaxing
effect of isoprenaline.
7. Effect on polymorphonuclear neutrophil granulocytes
Polymorphonuclear neutrophil granulocytes (PMN) are cells which are
mobilized during inflammatory phenomena and which can be stimulated
by various compounds, such as, for example,
formylmethionyl-leucyl-phenylalanine (FMLP) or prostaglandins E
(PGE.sub.1). The PMN granulocytes respond to these extracellular
stimuli with an activation of the oxygen metabolism with release of
toxic oxygenated metabolites. An excessive response of the PMN
granulocytes may be the cause of a painful inflammation and is also
accompanied by a reduction in the level of cyclic adenosine
monophosphate (cAMP) in these granulocytes. Consequently, compounds
which inhibit the respiratory burst of PMN granulocytes or which
increase the level of cAMP can be regarded as very important in the
treatment of arthritis and asthma. The aim of the pharmacological
test described below is to show that the compounds according to the
invention have a double character: on the one hand, they inhibit
the stimulation of PMN granulocytes, and on the other hand, they
increase the level of cAMP in these cells.
Inhibition of the stimulation of PMN granulocytes
The stimulation of PMN granulocytes is evidenced by the
chemiluminescence which accompanies the activation of the oxygen
metabolism when these cells are stimulated in the presence of
luminol (5-amino-2,3-dihydro-1,4-phthalazinedione).
Rat PMN granulocytes (5.times.10.sup.6 /ml) are preincubated in a
phosphate buffer (150 .mu.moles/liter, pH 7.4), containing luminol
at a concentration of 10.sup.-5 mole per liter, for 15 minutes at
37.degree. C. and then for 5 minutes in the presence of the
compound to be tested at a concentration of 10.sup.-6
mole/liter.
The reaction of stimulation of PMN granulocytes is initiated by the
addition of FMLP to the medium at a final concentration of
3.2.times.10.sup.-7 mole/liter. The luminescence which results from
the stimulation is measured by means of an LKB 1251 luminometer
according to the method of C. DAHLGREN and O. STENDAHL (Infection
and Immunology, 37, (1982), 34-39). An experimental cycle lasts 38
seconds. The reaction is repeated 9 times for each compound to be
tested and the average of the results obtained is calculated.
In Table X below, the mean percentage of residual chemiluminescence
calculated with respect to a control test, in the course of which
the PMN granulocytes are incubated and stimulated by FMLP in the
absence of the compound to be tested (100% chemiluminescence), is
given for the compounds according to the invention and for
theophylline (reference compound).
TABLE X ______________________________________ Inhibition of the
stimulation of PMN granulocytes Compound Residual chemiluminescence
(10.sup.-6 mole/l) (in %) ______________________________________ 1
47 2 68 3 39 4 45 5 74 6 55 7 54 9 82 10 56 11 44 12 36 13 79 14 51
15 70 16 42 theophylline 100
______________________________________
This Table shows that at the concentration of 10.sup.-6 mole/l at
which all the compounds according to the invention are tested,
theophylline is completely inactive. In contrast, it can be seen
that at this concentration, the compounds according to the
invention cause a significant reduction in the chemiluminescence
and thus induce a significant inhibition of the stimulation of PMN
granulocytes.
It has also been observed that a concentration 100 times higher
(10.sup.-4 mole/l) is needed to obtain a comparable effect with
theophylline (residual chemiluminescence of 65%).
Increase in the level of cAMP
Rat PMN granulocytes (10.sup.7 in 200 .mu.l) are incubated in a
phosphate buffer (150 .mu.moles/liter, pH 7.4) at 37.degree. C. for
3 minutes in the presence of the compound to be tested at a
concentration of 3.2.times.10.sup.-6 mole per liter, and of
PGE.sub.1 in a concentration of 10.sup.-6 mole/liter. The reaction
is then stopped by addition of 1 ml of propanol. After
centrifugation at 15,000 g for 3 minutes, the supernatant is
recovered and evaporated and the amount of cAMP in the residue is
determined by radioimmunological assay according to the process
recommended by the supplier of the reagent used for this purpose
(Amersham).
A control test is carried out under the same conditions, but in the
absence of the compound to be tested.
The amounts of cAMP (expressed in picomoles) obtained in the course
of these tests, in comparison with theophylline, which is also used
at a concentration of 3.2.times.10.sup.-6 mole/liter, are given in
Table XI below.
TABLE XI ______________________________________ Amount of cAMP
produced Compound (in picomoles per 10.sup.7 cells)
______________________________________ 1 21.0 3 23.2 4 17.7 5 11.6
6 14.6 7 15.8 11 16.2 12 16.8 theophylline 5.0 control 4.3
______________________________________
This Table shows that the compounds according to the invention are
more active than theophylline and considerably increase the level
of cAMP in PMN granulocytes stimulated by PGE.sub.1.
8. Toxicity
The toxicity of the compounds according to the invention has been
determined on male NMRI mice by means of the Irwin's test (S.
IRWIN, Psychopharmacologia, 13, (1968), 222-257).
Progressive doses of the compound to be tested are administered
intraperitoneally to groups of three mice until the lethal dose is
reached (dose which causes the death of two animals out of three
within 48 hours). The lethal dose observed for the compounds
according to the invention is given in Table XII below. This Table
shows that the compounds according to the invention have a very low
toxicity, in contrast to physostigmine.
TABLE XII ______________________________________ Lethal dose
Compound (in mg/kg) ______________________________________ 1 898 2
285 3 297.8 4 165 5 277.4 6 635.6 7 279.3 8 326 9 297.8 10 311.8 11
325.8 12 325.8 13 247.3 14 156.8 15 >498 16 194 17 281.5 18 277
19 175.6 20 185 21 263 22 295 23 156 24 825 25 99.7 26 201.5
physostigmine 0.82 ______________________________________
9. Posology and administration
The pharmaceutical compositions containing the compounds according
to the invention can be administered orally, parenterally or
rectally. The pharmaceutical compositions which can be used for
oral administration may be solid or liquid, for example in the form
of tablets (coated or non-coated), pills, dragees, gelatine
capsules, solutions, syrups and the like. The compositions which
can be used for parenteral administration are the pharmaceutical
forms known for this mode of administration, for example aqueous or
oily solutions, suspensions or emulsions.
For rectal administration, the compositions containing the
compounds according to the invention are generally in the form of
suppositories.
The pharmaceutical forms, such as injectable solutions, injectable
suspensions, tablets, drops, suppositories and the like, are
prepared by the methods currently used by pharmacists. The
pharmaceutical forms also comprise compositions which can deliver
the active product in a progressive manner. The compounds according
to the invention are mixed with a solid or liquid, non-toxic
pharmaceutically acceptable carrier, and optionally with a
dispersing agent, a disintegrating agent, a stabilizing agent and
the like. Sweetening agents, coloring agents and the like may also
be added, if appropriate. The percentage of active compound in the
pharmaceutical compositions can vary within very wide limits,
according to the patient and the mode of administration, in
particular according to the frequency of administration.
As regards the daily dosage, this can vary within a wide range of
dosage units, for example from 0.05 to 0.5 g of active compound,
depending upon the mode of administration. Thus, for example, it
can be from 0.1 to 0.5 g, preferably 0.1 g, one to several times
per day, if the compound is administered in the form of a
tablet.
A formulation for tablets is given below as a non-limiting example
of a composition containing a compound of the formula I, which can
be administered orally:
______________________________________ Compound 1 50 mg
Methylcellulose (Methocel K4M) 200 mg Dry lactose 154 mg Aerosil 5
mg Anhydrous citric acid 60 mg Talc 11 mg Magnesium stearate 20 mg
______________________________________
* * * * *